Shared Resources Core 2: Quantitative Proteomics Core

项目摘要 本杰明·加西亚博士领导的定量蛋白质组学核心将提供尖端的质谱学 向P01项目团队成员提供基于(MS)的蛋白质组学技术，以帮助阐明生化 人类癌症中涉及组蛋白和组蛋白样突变的机制。这些MS方法将 包括了解蛋白质表达和翻译后修饰(PTM)的定量实验 福尔马林固定的培养和原代细胞系、组织/肿瘤的组蛋白和非组蛋白的研究 石蜡包埋(FFPE)存档样本。尽管大多数表观遗传学和染色质的研究人员 生物学领域利用基于抗体的方法来表征组蛋白修饰，抗体有许多 技术问题(如表位封闭)，这混淆了分析。因此，质谱学 提供了一种更公正、更互补的方法，对蛋白质也更敏感、更准确 PTM分析。加西亚实验室已经开发了十多年的组蛋白PTM分析方法。这里 在这份提案中，我们计划继续扩展我们的蛋白质组学工具箱，使P01团队成员能够 快速鉴定500个组蛋白PTM位点，进行组蛋白组合的定量分析 修饰，并确定哪些致癌的组蛋白突变影响染色质结构。此外，我们 将探索在表观遗传驱动的人类中改变的下游蛋白质信号通路 癌症，因为转录图谱并不总是提供最准确的蛋白质表达图像。 好了！

Project Summary The Quantitative Proteomics core led by Dr. Benjamin Garcia will provide cutting-edge mass spectrometry (MS) based proteomics technologies to the P01 Project team members to help elucidate biochemical mechanisms involved in histone and histone-like mutations in human cancers. These MS approaches will include quantitative experiments to understand protein expression and post-translational modifications (PTMs) on histone and non-histone proteins from both cultured and primary cell lines, tissue/tumors and formalin fixed paraffin embedded (FFPE) archived samples. Although most researchers in the epigenetics and chromatin biology fields utilize antibody based methods for histone modification characterization, antibodies have many technical issues (such as epitope occlusion), which confound the analyses. Mass spectrometry therefore provides a more unbiased and complementary approach, that is also more sensitive and accurate for protein PTM analysis. The Garcia Lab has for over a decade now developed methods for histone PTM analyses. Here in this propose we plan to continue to expand our proteomics toolbox to allow P01 team members to be able to characterize 500 histone PTM sites in rapid fashion, perform quantitative analyses of histone combinatorial modifications, and determine which oncogenic histone mutations affect chromatin structure. Additionally, we will explore the downstream protein signaling pathways that are altered in epigenetically driven human cancers, as transcriptional profiles do not always provide the most accurate pictures of protein expression. !